The broad aim is to elucidate basic mechanisms in selected areas of thyroid research, and, where possible, to relate them to clinical thyroid disease. The major areas to be investigated are: 1) Donor and acceptor sites in thyroglobulin (Tg) - We plan to locate donor and acceptor sites for T4 and T3 in Tg, and the effect thereon of iodine content, using either a pulse-chase 125I-labeling procedure, or treatment with Na3HB4, followed by trypsinization, separation of fragments by HPLC, sequencing, and 125I-iodoamino acid analysis. 2) Specificity in tyrosyl iodination sites in Tg - To determine whether early tyrosyl iodination sites are determined primarily by thyroid peroxidase (TPO) or by the structure of Tg, low iodine Tg will be radioiodinated enzymatically with TPO and chemically with I3- to matched low levels of iodination. The tyr sites iodinated under the two conditions will be located by digestion with trypsin, separation of trypsin fragments by HPLC, 125I counting, and sequencing of labeled tryptic fragments. 3) Mechanism of TPO-catalyzed coupling and of its stimulation by diiodotyrosine (DIT) - We have obtained evidence suggesting that TPO- catalyzed coupling is a radical reaction. We have also shown that the coupling reaction is markedly stimulated by low concentrations of free DIT. We plan further studies on the mechanism of coupling and of DIT-stimulation by using cyt c peroxidase Compound ES, a stable complex that catalyzes coupling efficiently. We shall also measure thyroidal free DIT levels in various states of thyroid function to test the possible physiological significance of the DIT stimulation. 4) Source of H2O2 in the thyroid - We plan to study the particulate, Ca++- activated, H2O2 generating system in the thyroid in familial as well as other thyroid disorders, especially with reference to the cyt P450-cytP450 reductase system. 5) TPO structure and thyroid microsomal autoantibodies - We plan to prepare synthetic peptides representing defined regions of TPO for locating active sites and for mapping epitopes that are important in autoimmune thyroid disease.